The long-term goal of this project is to develop an understanding of the cellular and molecular mechanisms of human cytomegalovirus (HCMV) persistence in the host. HCMV is a significant pathogen in immune compromised patients and the leading viral cause of congenital defects. Our group and others have shown that HCMV and other herpesviruses encode multiple microRNAs (miRNAs) that are small 21-24 base pair (bp) single-stranded RNA species that regulate gene expression through post-transcriptional mechanisms. Herpesvirus miRNAs have been shown to target many different cellular and viral processes involved in viral immune recognition, apoptosis, cell cycle regulation, as well as viral latency and lytic replication. During the previous funding period our group has made significant advances elucidating functional roles for HCMV miRNAs. We have demonstrated that one of the HCMV miRNAs efficiently targets and reduces expression the IE72 protein that significantly reduces viral replication suggesting a role for maintenance of latent virus. Secondly, we have also found that the HCMV miRNAs coordinately work together to efficiently down-regulate individual genes that may explain why individual miRNA knockouts in the viral genome do not show phenotypic effects. Additionally, the HCMV miRNAs appear to also target multiple individual cellular genes in the same cellular pathways including two potential antiviral cytokines IL-1 and TNF-? as well as the NF?B inhibitor IkBa . Lastly, we have observed that a double HCMV miRNA mutation results in an increase in viral reactivation while two other viral miRNA double mutants fail to reactivate in a human progenitor cell (HPC) culture system in vitro. Interestingly, one of the HCMV miRNAs in the double-mutants that fail to reactivate targets two NF?B activators suggesting that NFkB activation is deleterious for reactivation of virus. Therefore, in the current proposal we will fully characterize the IL-1 and TNF-? signaling pathways targeted by the HCMV miRNAs and their functional relevance for viral latency and replication in a CD34+ HPC. We will also examine the function of HCMV miRNA targeting of the IL-1 and TNF-? signaling pathways during latency and reactivation in a newly developed human CD34+-engrafted NOD-scidIL2Rgc null mouse model that is able to support latent HCMV infection as well as reactivation from latency. We hypothesize that HCMV miRNA repression of viral replication is related to NFkB activation through the IL-1 and TNF-? pathways.